Several genes have been identified that are thought to play a role in regulating normal cell growth. A subset of these genes, termed ras, consists of at least three members, N-ras, H-ras, and K-ras2. Altered forms of ras, termed oncogenes, have been implicated as causative agent in cancer. Both the normal cellular genes, and the oncogenes encode chemically related proteins, generically referred to as p21.
Ras oncogenes, and their normal cellular counterparts, have been cloned and sequenced from a variety of species. Comparison of the structure of these two genes has revealed that they differ by point mutations that alter the amino acid sequence of the p21 protein. Naturally occurring mutations in the ras oncogenes have been identified in codons 12, 13, 59, and 61. In vitro mutagenesis work has shown that mutations in codon 63, 116, and 119 also result in transforming activity. The most frequently observed mutation which converts a normal cellular ras gene into its oncogenic counterpart is a substitution of gaycane at position 12 by any other amino acid residue, with the exception of proline. Transforming activity is also observed if glycine is deleted, or if amino acids are inserted between alanine at position 11 and glycine at position 12.
Mutations at position 61 also play an important role in the generation of ras oncogenes. Substitution of glutamine for any other amino acid, except proline or glummac acid in the cellular ras gene yields ras oncogenes with transforming activity.
In relation to normal cellular ras genes and their oncogenic counterparts, there are at least four known retrovital ras oncogenes which exhibit transforming activity. Unlike their non-retroviral analogues, the retroviral genes exhibit two mutations. The biologically significance of these double mutations is at present unclear.
Both the normal ras and oncogenic p21 proteins, regardless of their phylogenetic origin, bind guanine nucleotides, GTP and GDP, and possess intrinsic GTPase activity. Temeles et al., Nature, 313:700 (1985). The significance of these biochemical properties to the biological activities of the ras proteins has been demonstrated as follows: first, microinjection of anti-ras antibodies that interfere with guanine nucleotide binding reverses the malignant phenotype of NIH 3T3 cells transformed by ras oncogenes. Clark, et el., Proc. Natl. Acad. Sci. U.S.A., 82:5280 (1985); Feramisco, et al., Nature, 314:639 (1985). Second, ras oncogenic proteins that exhibit mutations which result in the inability of p21 to bind guanine nucleotides do not transform NIH 3T3 cells. Willumsen, et al., Mol. Cell. Biol., 6:2646 (1986). Third, some ras oncogenes produce p21 proteins that have much reduced GTPase activity compared to their normal cellular counterparts. The biological role of GTPase activity associated with either ras or its oncogenic counterpart remains unknown.
Recently a cytoplasmic factor has been identified which stimulates normal ras p21 GTPase activity, but does not effect GTPase activity associated with the oncogenic routants. M. Trahey and F. McCormick, Science, 238:542 (1987). The activity has been associated with a protein, termed GAP, which is the acronym for (GTPase activating protein. GAP is thought to be a cytoplasmic protein but is presumably capable of moving from the cytosol to the plasma membrane where it interacts with p21.
As alluded to above, ras oncogenes have been implicated in the development of a variety of tumors, and have been shown to be involved in about 10-40% of the most common forms of human cancer. H. Varmus, Annual Rev. Genetics, 18:553 (1984); M. Barbacid, in Important Advances in Oncology (1986), ed. B. DeVita, S. Helman, S. Rosenberg, pages 3-22, Philadelphia:Lippincott. For example, ras oneogenes have been consistenfly identified in carcinomas of the bladder, colon, kidney, liver, lung, ovary, pancreas and stomach. They also have been identified in hematopoietic tumors of lymphoid and myeloid lineage, as well as in tumors of mesenchymal origin. Furthermore, melanomas, teratocarcinomas, neuroblastomas, and gliomas have also been shown to possess ras oncogenes.
Considering the possible association of ras oncogenes and cancer, there has been considerable work focused on diagnostic tests for detecting the presence of the oncogene product, p21, or the mutant oncogenes. Early tests, which are still employed in many instances, identify the presence of ras oncogenes in transfection assays which identify p21 by its ability to transform NIH 3T3 cells. Lane, et at., Proc. Natl., Acad. Sci. U.S.A., 78:5185 (1981); and B. Shilo, and R. A. Weinberg, Nature, 289:607 (1981). This method is insensitive, laborious, and requires a skilled laboratory technician to perform adequately.
A second diagnostic method centers around oligonucleotide probes to identify single, point mutations in genomic DNA. This technique is based on the observation that hybrids between oligonucleotides form a perfect match with genomic sequences, that is, nonmutated genomic sequences are more stable than those that contain a single mismatch. The latter, of course, being a point mutation in p21 associated with the ras oncogenes. Although this technique is clearly more sensitive and easier to perform than the transfection assay, it is nevertheless also cumbersome to perform. This is because there are theoretically almost 100 base substitutions which can yield ras oncogenes. Thus, in order to be able to detect these substitutions multiple oligonucleotide probes must be employed containing each of the three possible substitutions at a particular residue. Bos, et al., Nature, 315:726 (1985); Valenzuela, et al., Nuc. Acid Res., 14:843 (1986).
In addition to the transfection and oligonucleotide assays, additional nucleic acid hybridization techniques have been developed to identify ras oncogenes. One such method is based on the unusual electrophoretic migration of DNA heteroduplexes containing single based mismatches in denaturing gradient gels. Myers et al., Nature, 313:495 (1985). This technique only detects between about 25-40% of all possible base substitutions, and requires a skilled technician to prepare the denaturing gradient gels. More sensitive techniques which are refinements of this technique are described by Winter, et al., Proc. Natl., Acad. Sci. U.S.A., 82:7575 (1985); and Myers, et al., Science, 230:1242 (1985).
Immunologic approaches have been taken to detect the product of the ras oncogenes. Antibodies, either polyclonal or monoclonal, have been generated against the intact ras oncogene p21, or against chemically synthesized peptides having sequences similar to oncogene p21, or the non-transforming counterpart. U.S. patent application Ser. No. 938,581; EP Patent Publication 108,564 to Cline et al.; Tamura, et al., Cell, 34:587 (1983); PCT Application WO/84/01389 to Weinberg et al. For the most part, unfortunately, antibodies have been disappointing as diagnostic tools with which to identify ras oncogenic p21 in human tissue sections. This is because either the antibodies that have been generated to date recognize the normal cellular ras protein as well as the oncogenic protein, or in those instances where a monocional antibody has been generated that specifically recognizes the oncogenic protein, it exhibits non-specific staining of tumor biopsies.
While ras oncogenic p21 is an effective tumorigenic agent, recent studies have shown that normal ras p21 can induce the malignant phenotype. Chang et al., Nature, 297:7479 (1982); Pulciani, et al., Mol. Cell. Biol., 5:2836 (1985). For example, transfection of normal H-ras DNA has been shown to induce malignant transformation. It is further noteworthy that normal ras gene amplification has been observed in several human tumors, and has an apparent incidence of about 1%. Pulciani, et al., above; Yokota, et al., Science, 231:261 (1986). The various diagnostic test used to detect ras oncogenes or oncogenic p21 have been applied to the detection of normal ras p21 with similar limited success.
It should be apparent from the foregoing that while there are a number of diagnostic methods for determining the presence of ras oncogenes, or their transforming proteins, there is still a need for fast and reliable diagnostic methods that will permit their routine identification.
By and large, the vast majority of cancer therapeutics function by killing dividing cells, and because of this lack of specificity, kill normal as well as cancer cells. Thus, despite knowledge of the existence of normal cellular ras genes, or their oncogenic counterparts, there have been identified few therapeutics that can interfere with, or reverse the transformed state that are not generally cytotoxic. Valeriote, F. and Putten, L., Cancer Res., 35:2619 (1975). The exceptions include anti-ras monoclonal antibody, Y13-259, which has been shown to selectively block the morphologic transformarion induced by oncogenic ras proteins. Mulcahy, et al., Nature, 313:241 (1985). Also, U.S. patent application Ser. No. 938,581 shows a monocional antibody directed against oncogenic p21 having serine at position 12. This antibody, when microinjected into ras transformed cells causes the cells to revert to a normal cell phenotype. It has no effect on normal cells. Unfortunately, because ras is located on the cytoplasmic side of the plasma membrane, where it interacts with GTP and GAP, large molecular weight molecules such as antibodies may not have immediate therapeutic significance in the clinical setting. Thus, a method that will facilitate the identification of cancer therapeutics is sorely needed.